Investigation into an efficient synthesis of 2,3-dehydro-N-acetyl neuraminic acid leads to three decarboxylated sialic acid dimers

Article abstract of DOI:10.1016/j.carres.2008.01.033

Sialic acid, an important carbohydrate found incorporated on the cell surface of many organisms, has been modified for use in a wide range of biological and pharmaceutical applications. We hypothesized that 4,7,8,9-tetra-O-acetyl-2-deoxy-2,3-dehydro-N-acetyl neuraminic acid methyl ester (4) could be efficiently synthesized in a one-pot reaction by heating peracetylated sialic acid (2) in pyridine and acetic anhydride to induce β-elimination. When reduced to practice, this reaction produced only modest yields of 4. Six compounds, including three new decarboxylated sialic acid dimers, were also found to have been synthesized in the reaction. In an effort to better understand the chemistry and the mechanisms of this reaction, all of the side products were isolated and fully characterized.

Full text of DOI:10.1016/j.carres.2008.01.033

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 936–940
Note
Investigation into an efficient synthesis of 2,3-dehydro-N-acetyl
neuraminic acid leads to three decarboxylated sialic acid dimers
Evan J. Horn, Jonel P. Saludes and Jacquelyn Gervay-Hague*
Department of Chemistry, University of California, Davis, One Shields Ave., Davis, CA 95616, USA
Received 7 November 2007; received in revised form 19 January 2008; accepted 24 January 2008
Available online 2 February 2008
Abstract—Sialic acid, an important carbohydrate found incorporated on the cell surface of many organisms, has been modified
for use in a wide range of biological and pharmaceutical applications. We hypothesized that 4,7,8,9-tetra-O-acetyl-2-deoxy-2,3-
dehydro-N-acetyl neuraminic acid methyl ester (4) could be efficiently synthesized in a one-pot reaction by heating peracetylated
sialic acid (2) in pyridine and acetic anhydride to induce b-elimination. When reduced to practice, this reaction produced only mod-
est yields of 4. Six compounds, including three new decarboxylated sialic acid dimers, were also found to have been synthesized in
the reaction. In an effort to better understand the chemistry and the mechanisms of this reaction, all of the side products were
isolated and fully characterized.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Sialic acid; Decarboxylation; Neu5Ac2en
Sialic acid (N-acetylneuraminic acid; Neu5Ac, 1) has
been modified for use in a wide range of biological
and pharmaceutical applications. The 2,3-dehydro
derivative of Neu5Ac (Neu5Ac2en, 4) is a key interme-
diate in the preparation of the commercial anti-influenza
drug, Zanamivir, which was found to be more effective
than adamantane-based compounds.1,2 Because of its
synthetic importance, several methods have been
reported for the synthesis of 4.3,4 These include the most
widely used b-elimination of peracetylated Neu5Ac
glycosyl chloride 35 catalyzed by Et3N or 1,8-diazabi-
cyclo[5.4.0]undec-7-ene (DBU) (Scheme 1),6,7 TMSOTf-
catalyzed elimination of peracetylated Neu5Ac methyl
ester 2,8 and oxidation9 or elimination10 of Neu5Ac
thioglycoside. A recent approach to the preparation of
4 from 3 utilized Na2HPO4 in refluxing acetonitrile for
3 h.11 Although this method gave an almost quantitative
yield, the preparation of 3 required several steps and
careful handling of HCl gas.
Our laboratory has found derivatives of 1 and 4 to be
useful building blocks for constructing amide-linked
homooligomers with stable secondary structures,12,13
and we typically employed DBU-catalyzed b-elimina-
tion to make 4. However, it was our hypothesis that
the C-2 acetate could be a sufficiently good leaving
group under the basic conditions of peracetylation,
and that by simply heating 2 in a one-pot technique,
we could induce b-elimination to afford 4. To test this
hypothesis, 1 was peracetylated to yield nearly pure
product as evidenced by 1H NMR and MS. The solution
was then heated to 100 °C for 4 h. The crude reaction
mixture was carried forward and treated with ethereal
diazomethane to methylate the carboxylic acid to facili-
tate purification (Scheme 2).
Initial 1H NMR analysis showed that several products
were formed in the reaction (Scheme 2), including com-
pound 4 (confirmed via a doublet at d 6.08, J = 1.8 Hz).
The mixture was purified by a series of chromatographic
procedures (Fig. S6, Supplementary data) to yield a
total of seven compounds, with 4 isolated in only 21%.
Two other products 5 (37%) and its anomer 6 (3%)
were quite unexpected, with both having undergone
*
Corresponding author. Tel.: +1 530 754 9577; fax: +1 530 752
8995; e-mail: gervay@chem.ucdavis.edu
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.01.033

E. J. Horn et al. / Carbohydrate Research 343 (2008) 936–940
937
1. Ac2O, Pyridine
OAc
OH
2. Dowex H+ resin
OH
HO
OAc
CO2Me
AcO
OH
OAc
MeOH
O
CO2H
O
AcHN
AcHN
3. CH2N2
HO
1
AcO
2
HCl(g)
AcCl
"one pot"
OAc
OAc
OAc
OAc
Cl
AcO
AcO
CO2Me
DBU, benzene
O
CO2Me
O
AcHN
AcHN
AcO
AcO
4
3
Scheme 1. The most widely used route to 4, and our proposed one-pot synthesis.
OAc
OAc
OAc
OAc
AcO
AcO
1. Ac2O, pyridine 24 h, rt
2. 100 oC, 4 h
3. Dowex H+ resin
4. CH2N2
CO2Me
OAc
O
H
O
AcHN
AcHN
1
AcO
5 (37%)
AcO
4 (21%)
OAc
OAc
NHAc
OAc
OAc
O
H
AcO
AcO
AcO
OAc
AcHN
OAc
NHAc
OAc
O
O
OAc
O
AcHN
O
OAc
AcO
AcO
OAc
OAc
OAc
O
6 (3%)
7 (2%)
O
O
8 (5%)
OAc
NHAc
OAc
OAc
OAc
OAc
AcO
OAc
AcO
OAc
AcO
OAc
NHAc
OAc
OAc
O
O
OAc
OAc
O
O
O
AcHN
AcHN
O
O
OAc
AcO
O
AcO
9 (15%)
Scheme 2. Products isolated from one-pot reaction.
10 (2%)
decarboxylation. Both 5 and 6 were previously synthe-
sized using 4:1 toluene–pyridine in the presence of
lead(IV) acetate.14 Formation of these compounds
under standard peracetylation conditions was unprece-
dented. Compound 7, a 1?7 lactone which is a common
side product of peracetylation,15 was also found in small
amounts (2%).
Compounds 8, 9, and 10 are novel compounds. We
first believed these to be dimers based on a few observa-
tions. Their mass spectra showed molecular ions close to
twice that of the previously isolated monomers, and the
number of acetate peaks in the 1H NMR spectra corres-
ponded to dimeric, peracetylated structures. In addition,
their 1H NMR spectra showed similarities to 2, 4, 5, and
6, leading us to believe that each of these dimers are
composed of a combination of any two of these previ-
ously isolated compounds. Compound 9, being the
dimer isolated in highest yield, was characterized first.
Its low-resolution mass of m/z 957.3 [M+Na]+ suggest
that it was composed of a combination of 2 and either
5 or 6. Deuterium exchange and 1H NMR studies
revealed two exchangeable protons from NHAc, indi-
cating that the dimer is connected via an ester linkage.
The mass difference of 74 amu between 9 and the sum
of 2 (m/z 533.2 [M]+) and 5 (or 6) (m/z 475.2 [M]+) indi-
cate the absence of an acetate and Me of the methyl
ester. Heteronuclear correlation by HSQC revealed that
1
the downfield H NMR peak at d 6.15 is anomeric (13C
NMR d 93.5), and not due to an olefinic proton. The
configuration of this anomeric center was determined

938
E. J. Horn et al. / Carbohydrate Research 343 (2008) 936–940
by applying J-based analysis using Karplus relationships
to the anomeric proton resonance. We found that
this proton is equatorial as shown by a doublet
(J = 1.8 Hz) that is due to the coupling with axial
methylene proton on C-2. If the anomeric H were axial,
a distinct doublet of doublets would have been observed
due to large trans diaxial and small equatorial–axial
couplings (J1,2ax = ꢀ10 Hz; J1,2eq = ꢀ2 Hz). This led
us to conclude that 9 is a b-linked dimer composed of
same peracetylation conditions shown in Scheme 1.
We only recovered 2 and none of 4–10. This suggests
that the free acid plays a role in the formation of the
observed products. In summary, our one-pot method
generated seven compounds, including Neu5Ac2en (4)
and three novel dimeric structures. Peracetylation of
sialic acid using pyridine and acetic anhydride is
common protecting group chemistry. It is surprising
that relatively high amounts of 2 underwent decarboxyl-
ation under typical peracetylation conditions and heat-
ing. We are further investigating the mechanism of
these reactions with the goal of directing the selectivity
to the formation of 4.
1
2 and 5. The remaining H and 13C NMR resonances
were assigned using a combination of COSY, HSQC,
and HMBC (Table 2, Supplementary data). The key
connectivity between 2 and 5 that completed the struc-
tural characterization of 9 was done by HMBC correla-
tions (J = 8 Hz) between H-30 and C-10 and between
C-10 and H-1 (Fig. 1).
1. Experimental
The structures of 8 and 10 were solved in the same
manner as 9. Compound 10 has the same mass and
similar H NMR spectra as 9. The key difference is the
1.1. General methods
1
resonance at d 5.65 (dd, J = 10.2, 1.8 Hz) due to axial
anomeric proton indicating that 10 is an a-linked dimer
composed of 2 and 6. The connectivity was confirmed by
HMBC correlations (J = 8 Hz) as shown in Figure 1.
The NMR spectra of 8 clearly indicate that it possesses
the same olefinic system as 4 (1H NMR d 6.04; 13C
NMR d 110.0) and the same equatorial anomeric proton
as 9 (d 6.38, d, J = 1.8 Hz). The low-resolution mass of
m/z 897.2 [M+Na]+ that is 74 amu less than the sum of
4 and 5 is equivalent to the absence of a Me and an ace-
tate. Deuterium exchange studies also revealed two
exchangeable protons from NHAc indicating an ester
NMR spectra were recorded on Varian 300 and
600 MHz NMR spectrometers. Chemical shifts were
referenced to residual CHCl3 (dH = 7.26) and CDCl3
(dC = 77.1). Low-resolution mass spectra were obtained
by electrospray ionization using Finnigan Mat LCQ-
Deca mass spectrometer. High resolution mass spectra
were obtained using Applied Biosystems 4700 MAL-
DI-TOF with internal calibration. Reactions were
monitored by TLC using Si Gel 60 F254 glass-backed
plates (Merck). Reverse phase HPLC was performed
using Waters 600 on a C18 column (250 ꢁ 10 mm,
7 lm particle size, Vydac) at 90 min elution time using
a gradient of 5–50% MeOH in H2O at 4 mL/min flow
rate with detection set at 215 and 225 nm.
1
linkage. The remaining H and 13C NMR resonances
were assigned (Table 3, Supplementary data), the b-link-
age between 4 and 5 determined by J-based analysis on
the anomeric proton (d 6.38, d, J = 1.8 Hz) and their
connectivity was confirmed by HMBC correlations
(J = 5 Hz) as shown in Figure 1.
1.2. General synthesis
Pyridine (7.5 mL) and Ac2O (7.5 mL) were added to
sialic acid (500 mg, 1.6 mmol) under N2 at rt. The
suspension was stirred and the reaction was complete
As an initial step in our investigation of the reaction
mechanism, the methyl ester of 1 was subjected to the
OAc
9'
OAc
AcHN8'
7' OAc
OAc
OAc
NHAc
5'
NHAc
4
6'
3
2
3
OAc
AcO
4
OAc
AcO
4'
O
2
7' OAc
9'
6'
OAc
5 OAc
8'
5
OAc
OAc
OAc
OAc
1' O
1
O
2'
O
1
1'
AcHN
H
4'
3'
6
7
7
5'
O
O
6
O
H
O
8
H
8
AcO
8
H
9
OAc
OAc
OAc
AcO
2
3
9'
7'OAc
6'
4
NHAc
8'
O
O
O
2'
3'
1
1'
7
5
6
AcHN
4'
5'
8
O
H
OAc
AcO
AcO
OAc
H
10
Figure 1. Key HMBC correlations in determining the connectivity for 8, 9, and 10.

E. J. Horn et al. / Carbohydrate Research 343 (2008) 936–940
939
after 12 h as observed by TLC, resulting in full pera-
cetylation (Rf = 0.4 in 65:15:8 CHCl3–MeOH–H2O). A
small aliquot was removed from the solution, diluted
with EtOAc, and washed with 1 N HCl. The organic
NH peaks at 5.50 and 6.71 ppm was observed. A cou-
pling constant of 5 Hz was used to observe the HMBC
correlations between C-10 and H-1 and H-30. All other
1
HMBC correlations were observed at 8 Hz. H NMR
1
layer was concentrated to dryness, the H NMR spec-
(600 MHz, CDCl3): d 6.72 (d, 1H, J = 9.6 Hz), 6.38 (d,
1H, J = 1.8 Hz), 6.04 (d, 1H, J = 2.4 Hz), 5.74 (dd,
1H, J = 8.8, 2.4 Hz), 5.50 (m, 3H), 5.37 (m, 2H), 5.09
(m, 1H), 4.47 (m, 2H), 4.37 (dd, 1H, J = 12.6, 2.4 Hz),
4.20 (m, 3H), 4.05 (dd, 1H, J = 12.6, 7.2 Hz), 3.97 (dd,
1H, J = 10.2, 1.8 Hz), 2.25 (dd, 1H, J = 12.4, 4.4 Hz),
2.18–1.89 (m, 32H). 13C NMR (150 MHz, CDCl3): d
171.3, 171.2, 171.1, 170.9, 170.7, 170.5, 170.4, 170.3,
170.0, 159.2, 145.1, 110.0, 92.2, 75.5, 71.8, 71.3, 69.5,
68.6, 68.3, 68.0, 66.5, 62.5, 61.5, 49.0, 47.4, 31.8, 23.3,
23.1, 21.2, 20.9, 20.8. HRMS m/z: calcd for
C37H50N2NaO22, 897.2753; found, 897.2790.
trum recorded, and was found to be consistent with
the literature16 for 2. The solution was heated to
100 °C for 4 h, at which time 2 had disappeared by
TLC. The mixture was cooled to rt and concentrated.
The crude mixture was dissolved in MeOH, treated with
Dowex 50WX8-100 cationic resin for 30 min, filtered,
and concentrated. The brown glassy oil was dissolved
in 2:1 MeOH/Et2O and followed by dropwise addition
of freshly prepared CH2N2. A slight excess of CH2N2
was added to ensure complete methylation, and the
reaction was quenched with HOAc. The mixture was
concentrated to dryness in vacuo to yield a brownish
glassy oil (710 mg). The crude reaction mixture was
purified according to Figure S6, Supplementary data,
to yield 4, 5, 6, 7, 8, 9, and 10.
1.2.6.
penta-O-acetyl-N-acetyl
4-Acetamido-3,6,7,8-tetra-O-acetyl-1-[1,4,7,8,9-
neuraminyl]-2,4-dideoxy-b-D-
galacto-octopyranose (9). Isolated yield is 109 mg
1
(15%) as a yellow glassy oil. HPLC tR = 84.4 min. H
1.2.1. 4,7,8,9-Tetra-O-acetyl-2-deoxy-2,3-dehydro-N-acetyl
neuraminic acid methyl ester (4). Isolated yield is
NMR spectrum was also recorded in CDCl3 with 3 drops
of D2O added. After shaking, the mixture was allowed to
sit overnight, H NMR spectrum was recorded and the
1
1
158 mg (23%) as a yellow oil. HPLC tR = 66.6 min. H
and 13C NMR are consistent with the literature.7 LRMS
disappearance of amide NH peaks at 5.51 and
6.92 ppm was observed. The product was slightly unsta-
ble at room temperature at both dry and as CDCl3
solution, and was found to decompose to the 2 monomer
units. All HMBC correlations were recorded using a cou-
m/z: calcd for C20H27NO12Na+, 496.1; found, 496.2.
1.2.2. 4-Acetamido-1,3,6,7,8-penta-O-acetyl-2-4-dideoxy-
b-D-galacto-octopyranose (5). Isolated yield is 292 mg
1
1
(37%) as a yellow glassy oil. HPLC tR = 53.8 min. H
pling constant of 8 Hz. H NMR (600 MHz, CDCl3): d
and 13C NMR spectra are consistent with the litera-
ture.14 LRMS m/z: calcd for C20H29NO12Na+, 498.2;
found, 498.2.
6.92 (d, 1H, J = 12.0 Hz), 6.15 (d, 1H, J = 1.8 Hz),
5.51 (dd, 1H, J = 7.8, 2.4 Hz), 5.48 (t, 1H, 1.8 Hz), 5.44
(dt, 1H, J = 4.2, 2.4 Hz), 5.40 (d, 1H, J = 4.8 Hz), 5.36
(d, 1H, J = 4.8 Hz), 5.35 (d, 1H, J = 10.2 Hz), 5.17 (m,
1H), 4.48 (dd, 1H, J = 12.6, 2.4 Hz), 4.44 (dd, 1H,
J = 12.6, 3.0 Hz), 4.30 (dd, 1H, J = 10.8, 1.2 Hz), 4.19
(m, 4H), 4.04 (dd, 1H, J = 10.8, 2.4 Hz), 2.51 (dd, 1H,
J = 13.2, 4.8 Hz), 2.23 (ddd, 1H, J = 13.2, 4.2, 1.8 Hz),
2.15–1.89 (m, 35H). 13C NMR (150 MHz, CDCl3):
d171.6, 171.2, 171.1, 170.7, 170.6, 170.4, 170.3, 169.0,
165.2, 97.1, 93.5, 72.8, 72.5, 72.2, 70.7, 68.8, 68.3, 68.1,
66.5, 62.5, 61.7, 49.4, 49.1, 37.6, 36.7, 23.3, 21.3, 21.2,
21.1, 21.0, 20.9, 20.6, 20.1. HRMS m/z: calcd for
C39H54N2NaO24, 957.2964; found, 957.2990.
1.2.3. 4-Acetamido-1,3,6,7,8-penta-O-acetyl-2-4-dideoxy-
a-D-galacto-octopyranose (6). Isolated yield is 21 mg
1
(3%) as a yellow glassy oil. HPLC tR = 61.6 min. H
and 13C NMR spectra are consistent with the litera-
ture.14 LRMS m/z: calcd for C20H29NO12Na+, 498.2;
found, 498.2.
1.2.4. 5-Acetamido-2,4,8,9-tetra-O-acetyl-b-D-glycero-D-
galacto-2-nonulopyranosonic acid-1,7-lactone (7). Iso-
lated yield is 18 mg (2%) as a yellow glassy oil. HPLC
1
tR = 50.9 min. H and 13C NMR spectra were found
to be consistent with the literature.15 LRMS m/z: calcd
1.2.7. 4-Acetamido-3,6,7,8-tetra-O-acetyl-1-[1,4,7,8,9-
penta-O-acetyl-N-acetyl neuraminyl]-2,4-dideoxy-a-D-
for C19H25NO12Na+, 482.1; found, 482.1.
galacto-octopyranose (10). Isolated yield is 13 mg
1
1.2.5. 4-Acetamido-3,6,7,8-tetra-O-acetyl-1-[4,7,8,9-tetra-
O-acetyl-2-deoxy-2,3-dehydro-N-acetyl neuraminyl]-2,4-
dideoxy-b-D-galacto-octopyranose (8). Isolated yield is
32 mg (5%) as a colorless glassy oil. HPLC tR =
83.7 min. 1H NMR spectrum was also recorded in
CDCl3 with 3 drops of D2O added. After shaking, the
(1%) as a yellow glassy oil. HPLC tR = 73.7 min. H
NMR spectrum was also recorded in CDCl3 with 3
drops of D2O added. After shaking, the mixture was
allowed to sit overnight, 1H NMR spectrum was
recorded and the disappearance of amide NH peaks at
5.31 and 5.26 ppm was observed. The product was
slightly unstable at room temperature at both dry and
as CDCl3 solution, and was found to decompose into
1
mixture was allowed to sit overnight, H NMR spec-
trum was recorded and the disappearance of amide

940
E. J. Horn et al. / Carbohydrate Research 343 (2008) 936–940
the 2 monomer units, as well as epimerize to 7 in CDCl3.
References
All HMBC correlations were recorded using a coupling
constant of 8 Hz. H NMR (600 MHz, CDCl3): d 5.65
1
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(dd, 1H, J = 10.2, 1.8 Hz), 5.43 (dd, 1H, J = 7.2,
2.4 Hz), 5.37 (dd, 1H, J = 4.2, 2.4 Hz), 5.30 (m, 3H),
5.08 (m, 2H), 4.96 (m, 1H), 4.47 (dd, 1H, J = 12.6,
3.0 Hz), 4.30 (dd, 1H, J = 12.6, 2.4 Hz), 4.06 (m, 4H),
3.80 (dd, 1H, J = 10.2, 2.4 Hz), 2.48 (dd, 1H, J = 13.8,
4.8 Hz), 2.36 (ddd, 1H, J = 12.6, 4.8, 1.8 Hz), 2.14–
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Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.01.033.